Microbiome convergence enables siderophore-secreting-rhizobacteria to improve iron nutrition and yield of peanut intercropped with maize

Intercropping has the potential to improve plant nutrition as well as crop yield. However, the exact mechanism promoting improved nutrient acquisition and the role the rhizosphere microbiome may play in this process remains poorly understood. Here, we use a peanut/maize intercropping system to investigate the role of root-associated microbiota in iron nutrition in these crops, combining microbiome profiling, strain and substance isolation and functional validation. We find that intercropping increases iron nutrition in peanut but not in maize plants and that the microbiota composition changes and converges between the two plants tested in intercropping experiments. We identify a Pseudomonas secreted siderophore, pyoverdine, that improves iron nutrition in glasshouse and field experiments. Our results suggest that the presence of siderophore-secreting Pseudomonas in peanut and maize intercropped plays an important role in iron nutrition. These findings could be used to envision future intercropping practices aiming to improve plant nutrition.

L165-170.The authors found their strain Pseudomonas 1502IPR-01 similar to P. extremorientalis based on complete genome sequences.Later the authors named their Pseudomonas 1502IPR-01 strain as P. extremorientalis 1502IPR-01.This is not correct giving a taxonomy based on this comparison.The authors should be careful and mention that their Pseudomonas 1502IPR-01 is similar to P. extremorientalis throughout the paper and not call it P. extremorientalis 1502IPR-01.L197-200: What about competition between microbes and maize for siderophore?Material and Methods: Soil chemical factors are not complete in Supplementary Table 8.Total N is not enough.Nitrogen forms like NH4, NO3 should be provided.They play important role in microbes, and microbes interaction with plants.They might have indirect effect on the microbes producing siderophores.
In addition, organic carbon should be provided.
L 477-479: give the PCR conditions or the reference L767-769: For PCoA and Bray-Curtis, what type of data were used?Relative abundance or normalized data?How the data was normalized?Add information.
Reviewer #2: Remarks to the Author: This study is probably the most bottom-up approach to elucidate the mechanisms involved in the beneficial actions at work in the interactions between crops in intercropping.The manuscript details first how the maize-peanut intercropping (IM) improves the iron nutrition of peanut (a strategy I plant).The involvement of microbiome in this effect is validated and the authors show that intercropping causes shifts in the microbiome composition.They further analyzed the microbiome composition using 16S sequencing and identified several beneficial genera and Pseudomonas in particular.One Pseudomonas species was identified after a phylogenetic analysis as being P. extremeorientalis, which whows high siderophore production.Whole genome sequencing allowed the identification of genes involved in pyoverdine siderophore biosynthesis and uptake.Further, the pyoverdine from P. extremeorientalis was purified and its structure determined.Pot and field experiments revealed that P. extremeorientalis and its purified pyoverdine could improve the iron nutrition.
The manuscript is very well written and the figures are very instructive.The discussion is very interesting and suggests the involvement of other bacterial genera in the positive interaction that deserve further investigations.The data presented in supplementary figures complete nicely those presented in the main manuscript.

I have however few remarks:
-The use of a mutant of P. aeruginosa unable to produce any siderophore confirms the importance of pyoverdines whatever the differences in peptide chain in the beneficial effects observed, but a siderophore negative mutant of P. extreorientalis should have been constructed as well if possible.-In the same line: some genes involved in the synthesis of the pyoverdine peptide chain (NRPS) are presented, but it would be nice to present the entire locus, including pvdL (chromophore biosynthesis), pvdS (ECF sigma), fpvA (receptor) and other genes involved in uptake and synthesis as a supplementary figure .-Pseudomonas often produce a second siderophore next to pyoverdine.Is it the case for P. extremeorientalis?This is not mentioned in the manuscript.If it is the case it could justify why the construction of a siderophore negative mutant is difficult.
-Likewise, rhizosphere bacteria, especially pseudomonads, also produce antibiotic compounds able to tackle phytopathogens.Were any found in the genome?

Point-by-point responses to the Reviewers' Comments: Reviewer #1: General comments
The authors showed in this study that intercropping peanuts and maize has positive effect on iron acquisition in plants, particularly in peanuts.By carrying out experiments in greenhouse with mixed crops (peanuts and maize) and monoculture (peanuts or maize) with gamma irradiated soil or not, they showed that improved iron nutrition is facilitated by Pseudomonas.The authors have done extensive experiments, and Pseudomonas strain isolation and characterization (genome sequencing, siderophore production, siderophore types, et).They concluded that their Pseudomonas 1502IPR-01 strain secrete pyoverdine, what becomes the preferred route of iron acquisition, benefiting both peanut and maize plants.The role of Pseudomonas as PGPB is well known; besides traits of siderophore production, they are also capable of solubilize phosphate, produce hormones, secondary metabolites, etc.There are many literature available including on intercropping systems with grass and leguminous crops.Specific comments: Results: Comment 1: L135-144: This network analysis is just a correlation between abundances and do not reflect any ecological relationship between microbes or plant-microbe-plant as mentioned in the abstract.In addition, it also does not exclude the relationship between microbes because of a third factor.Thus, I suggest to delete this result from the manuscript.Response 1: Thank you for raising this point.We agree that network analysis is not so important to figure out the key microbiome members involved in iron nutrition improvement by intercropping.In this context, the results of the LefSe analysis, the relative abundance of biomarkers in the different rhizosphere and their correlation with soil-plant iron nutrition are much more relevant.We have deleted the network analysis from original Fig. 2c,d, Supplementary Fig. 8, Supplementary Table 2 and related figure legends, results, discussion, methods and references.
Comment 2: L165-170.The authors found their strain Pseudomonas 1502IPR-01 similar to P. extremorientalis based on complete genome sequences.Later the authors named their Pseudomonas 1502IPR-01 strain as P. extremorientalis 1502IPR-01.This is not correct giving a taxonomy based on this comparison.The authors should be careful and mention that their Pseudomonas 1502IPR-01 is similar to P. extremorientalis throughout the paper and not call it P. extremorientalis 1502IPR-01.Response 2: We agree with this suggestion.Now we refer to this strain as Pseudomonas sp.1502IPR-01 throughout the manuscript.
Comment 3: L197-200: What about competition between microbes and maize for siderophore?Response 3: Thank you for raising this important point.We agree that plants and microbes may compete for iron.Clearly, bacteria like Pseudomonas spp.primarily secrete pyoverdines to obtain iron for themselves and not to feed plants.However, siderophores like pyoverdines are highly diffusible such that there is always a certain loss of molecules (i.e., many iron-loaded molecules will not find their way back to bacterial cells), which then become available for plants (Kümmerli, 2023).Moreover, plants secrete nutrient-rich exudates that attract bacteria, potentially fostering a mutually beneficial nutrient vs. siderophore exchange (Kümmerli, 2023;Gu et al., 2020;Harbort et al., 2020;Sasse et al., 2018;Shirley et al., 2011).For these reasons, we believe that there is relatively little competition for iron between plants and bacteria.8. Total N is not enough.Nitrogen forms like NH4, NO3 should be provided.They play important role in microbes, and microbe interaction with plants.They might have indirect effect on the microbes producing siderophores.In addition, organic carbon should be provided.

Gu
Response 4: As requested, we have added the data on NH4 + -N, NO3 --N and organic carbon contents in Supplementary Table 7.
Comment 5: L 477-479: give the PCR conditions or the reference Response 5: Thank you for your suggestion.We have added the PCR conditions in lines 496-500 of the methods section in manuscript file with the tracked changes.
Comment 6: L767-769: For PCoA and Bray-Curtis, what type of data were used?Relative abundance or normalized data?How the data was normalized?Add information.
Response 6: For PCoA and Bray-Curits analysis, relative abundance was used without normalization.We have added the information in the legend of Fig. 2 in lines 1000-1010 in manuscript file with the tracked changes

Reviewer #2 (Remarks to the Author):
This study is probably the most bottom-up approach to elucidate the mechanisms involved in the beneficial actions at work in the interactions between crops in intercropping.The manuscript details first how the maize-peanut intercropping (IM) improves the iron nutrition of peanut (a strategy I plant).The involvement of microbiome in this effect is validated and the authors show that intercropping causes shifts in the microbiome composition.They further analyzed the microbiome composition using 16S sequencing and identified several beneficial genera and Pseudomonas in particular.One Pseudomonas species was identified after a phylogenetic analysis as being P. extremeorientalis, which whows high siderophore production.Whole genome sequencing allowed the identification of genes involved in pyoverdine siderophore biosynthesis and uptake.Further, the pyoverdine from P. extremeorientalis was purified and its structure determined.Pot and field experiments revealed that P. extremeorientalis and its purified pyoverdine could improve the iron nutrition.
The manuscript is very well written and the figures are very instructive.The discussion is very interesting and suggests the involvement of other bacterial genera in the positive interaction that deserve further investigations.The data presented in supplementary figures complete nicely those presented in the main manuscript.
I have however few remarks: Comment 7: The use of a mutant of P. aeruginosa unable to produce any siderophore confirms the importance of pyoverdines whatever the differences in peptide chain in the beneficial effects observed, but a siderophore negative mutant of P. extreorientalis should have been constructed as well if possible.
Response 7: We agree that experiments with a pyoverdine null mutant in the Pseudomonas sp.1502IPR-01 background would be the most appropriate way to provide direct genetic evidence for the effect of pyoverdine.That is the reason why we started to construct such a pyoverdine mutant back in 2020.Unfortunately, we were not successful, and it turned out to be impossible (for us) to genetically modify this natural isolate.Therefore, we had no other choice than to use the PAO1 pyoverdine null mutant as an alternative.P. aeruginosa isolates are also frequently found in soil and freshwater habitats, and this species has been used as a valid control in other studies on plant iron nutrition improvement (Hernandez-Calderon et al., 2018).It is a generalist species, thriving in many habitats and that is the reason why we believe that a P. aeruginosa pyoverdine null mutant is a valid alternative to provide the first genetic evidence that pyoverdine is involved in iron nutrition improvement.Comment 8: In the same line: some genes involved in the synthesis of the pyoverdine peptide chain (NRPS) are presented, but it would be nice to present the entire locus, including pvdL (chromophore biosynthesis), pvdS (ECF sigma), fpvA (receptor) and other genes involved in uptake and synthesis as a supplementary figure.

Hernandez
Response 8: Thank you for your suggestion.We now present the entire locus including pvdL (chromophore biosynthesis), pvdS (ECF sigma), fpvA (receptor) and other genes involved in iron uptake and synthesis.Some genes found in PAO1 have no homologues in Pseudomonas sp.1502IPR-01 and are therefore not shown in the figure.We think that this result is very important for the main message of the paper and that is why we included the new scheme in Fig. 3e and not in a supplementary figure.We have further updated the Supplementary Table 3 and implemented small edits to improve clarity.
Comment 9: Pseudomonas often produce a second siderophore next to pyoverdine.Is it the case for P. extremeorientalis?This is not mentioned in the manuscript.If it is the case it could justify why the construction of a siderophore negative mutant is difficult.
Response 9: Thank you for raising this important issue.It is correct that certain Pseudomonas strains can produce a secondary siderophore, but it is also common that strains only produce pyoverdine.Pyochelin, enantio-pyochelin, quinolobactin, thioquinolobactin, achromobactin, PDTC (pyridine-2,6-bis(thiocarboxylic acid)), yersiniabactin and pseudomonine are such secondary siderophores (Kümmerli, 2023).That is the reason why we blasted the homologous genes biosynthesizing those siderophores against the genome of Pseudomonas 1502IP1-01.There were no hits (Table R1).Moreover, there is no report in the literature about the ability of P. extremorientalis to produce a secondary siderophore.Finally, our own data show that pyoverdine explains 88.81-91.96% of the iron-binding capacity of this species (Supplementary Table 4).These pieces of evidence together suggest that Pseudomonas 1502IP1-01 has no secondary siderophore and if it has one it is not particularly relevant for iron scavenging.Comment 10: Likewise, rhizosphere bacteria, especially pseudomonads, also produce antibiotic compounds able to tackle phytopathogens.Were any found in the genome?Response 10: Thank you for your suggestion.We used antiSMASH 7.0.1 to predict the secondary metabolites of Pseudomonas 1502IPR-01.We found that Pseudomonas 1502IPR-01 genome has a gene cluster sharing 68% similarity with the viscosin biosynthetic gene cluster from Pseudomonas fluorescens SBW25 (Fig. R1), suggesting that Pseudomonas 1502IPR-01 may produce viscosin, a lipopeptide biosurfactant.
Many studies have reported that lipopeptides have antibacterial and antifungal properties.Viscosin and viscosin-like lipopeptides inhibit phytopathogens, like Rhizoctonia solani AG2-2(Oni et al., 2020), Pythium myriotylum CMR1(Oni et al., 2020) but also human pathogens like Methicillin-resistant Staphylococcus aureus (Syaban et al., 2021).While the presence of a viscosin synthesis cluster is interesting, we prefer to not report it in the main manuscript.This is because our paper is already quite dense and viscosin is not the focus of our study.Reviewers' Comments: Reviewer #1: Remarks to the Author: The authors have addressed most of my comments and questions but there is a major concern about PCoA and Bray-Curtis analyses.It is not correct as it is presented in Figure 2 -results based on non-normalized data.Normalization is necessary to equal the sequencing depth for PCoA and Bray-Curtis analyses.This can be done in different ways and one of them is to perform rarefaction to make a fair comparison between samples.In addition, why the authors deleted from M&M the R package used for PCoA and Brays-Curtis (lines 800-801) if they present their results in Figure 2?
The authors have determined NH4, NO3 and organic carbon contents (Supplementary Table 7) but do not discuss the nutrient stage of the soils impact on the Fe uptake and Pseudomonas.
Minor comments: The authors have not determined the ecological interactions between plant-microbe.Ecological interactions need mathematical models.Therefore, delete "through a complex plant-microbe-plant ecological interaction" Line 36 of the Abstract.
Page 17 delete line 361" Our findings offer novel biological insights into belowground ecological interaction networks."-this sentence misleads the readers.The authors have not determined the belowground ecological interactions.
Page 17, delete line 335-336 "Overall, our work highlights that intercropping holds high potential for a more sustainable and more ecological agriculture" Page 757: NH4OAc-K?
Reviewer #2: Remarks to the Author: The manuscript has been revised according to the suggestions made by the two reviewers.The rebuttal letter is well written and to the point (which is not always the case).For my part I was quite satisfied with the addition of the complete pyoverdine genes cluster in figure as I suggested.

Point-by-point responses to the Reviewers' Comments:
Reviewer #1: Comment 1: The authors have addressed most of my comments and questions but there is a major concern about PCoA and Bray-Curtis analyses.It is not correct as it is presented in Figure 2 -results based on non-normalized data.Normalization is necessary to equal the sequencing depth for PCoA and Bray-Curtis analyses.This can be done in different ways and one of them is to perform rarefaction to make a fair comparison between samples.In addition, why the authors deleted from M&M the R package used for PCoA and Brays-Curtis (lines 800-801) if they present their results in Figure 2? Response 1: Thank you for your advice.Important to note is that all results involving amplicon sequences are based on normalized data.We normalized the sequencing depth to 17,063 counts per sample using scaling with ranked subsampling (SRS) (Beule & Karlovsky, 2020).We are sorry for the incorrect response to comment 6 in the previous revision, which happened because we misunderstood your comment 6.We thought that you asked if we performed other normalization except rarefaction for PCoA and Brays-Curtis.The related information was described in lines 518-526.We choose SRS rather than the traditional rarefying method (random subsampling without replacement) because of the following reasons.1) rarefying has poor reproducibility and can lead to distortion of the community structure.2) SRS preserves the original community structure by minimizing subsampling errors.3) SRS method is increasingly used in the field because of its good performance, including in some high-level articles published in Nature Communications (Maëva et al., 2023), Global Change Biology (Julia et al., 2023) and New Phytologist (Camille et al., 2023).Furthermore, we did not delete the R package used for PcoA and Brays-Curtis from the methods.Instead, the related description has been moved to lines 523-526 in the Methods.In this way, the upstream and downstream analysis methods of the amplicon sequence are described together in one section, which is convenient for the reader.

Fig. R1
Fig. R1 Pseudomonas 1502IPR-01 may produce viscosin according to genome information.a organization of gene cluster in location of 2,588,401-2,695,974 nt in Pseudomonas 1502IPR-01 genome.b Blast result implies this gene cluster shares 68% similarity with the viscosin biosynthetic gene cluster.

Table R1
Pseudomonas sp.1502IPR-01 do not secrete secondary siderophores according to genomic search